A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods.The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.PCR is a powerful tool for the amplification of minimal amounts of initial target genetic sequences [1,2], and it has been widely used for quantitative analysis of nucleic acids in medical diagnostics, drug discovery, virus detection, environmental monitoring, gene expression analysis and the detection of genetically modification organisms (GMO) [3-5]. Fluorescent quantitative PCR (FQ-PCR) can quantify initial target molecules by detecting the amplicon accumulation with fluorescently-tagged probes at each reaction cycle. Because of its ease of use, high throughput ability, decreased post-PCR manipulation, and lack of cross-contamination of PCR amplicons, FQ-PCR method is becoming the new gold standard met